Purification of pharmaceutical grade monoclonal antibody protein from cell culture media includes harvest/clarification followed by purification by using a series of column chromatography steps in combination with membrane ultrafiltration and diafiltration. After purification, the desired antibody preparation is suitably formulated and stored in appropriate conditions. However, many times, these steps do not provide the antibody with the desired level of purity and quality that are required for their pharmaceutical use. Sometimes, process-related and product-related impurities are observed to co-elute with the desired antibody during column purification. Therefore, it is important to reduce or remove such impurities from the desired preparation. Moreover, protein aggregation is a major concern during monoclonal antibody (mAb) production. The presence of aggregates can reduce the therapeutic efficacy of monoclonal antibody and known to trigger immunogenic responses in humans. Therefore, it is necessary to remove aggregates from the desired preparation of monoclonal antibody during downstream purification, mainly by column chromatography. With an aim to resolve this problem, the present invention provides novel method of purification of antibodies, which helps in the removal of process- and product-related impurities along with high molecular weight aggregates up to the desired level from a cell-free culture medium containing the antibody of interest by using a series of column chromatography in a particular manner. In the current invention, the said process of purification of antibody demonstrates well-controlled process of purification in a straight-forward manner which yields to a highly purified preparation of antibody with more than 80% recovery. In the current invention, the highly purified preparation of an antibody means a preparation of antibody with at least 99% purity and substantially free of process- and product related impurities and essentially devoid of high molecular weight aggregates of protein. Furthermore, the current invention provides a highly scalable and reproducible process of purification of monoclonal antibody. The described novel process of purification provides a common platform for purification of various antibodies for therapeutic use, in terms of process economy and industry viability.
Some of such techniques are disclosed in following patents:
U.S. Pat. No. 6,417,335 discloses method for purifying an antibody from a composition comprising the antibody and a contaminant, which method comprises: (a) loading the composition onto a cation exchange resin, wherein the amount of antibody loaded onto the cation exchange resin is from about 20 mg to about 35 mg of the antibody per mL of cation exchange resin; and (b) eluting the antibody from the cation exchange resin.
U.S. Pat. No. 7,863,426 describes method for producing a host cell protein-(HCP) reduced antibody preparation from a mixture comprising an antibody and at least one HCP, comprising an ion exchange separation step wherein the mixture is subjected to a first ion exchange material, such that the HCP-reduced antibody preparation is obtained.
Other relevant patents of the present field of invention are U.S. Pat. No. 6,489,447 EP 1075488; EP1308455; EP1308456B etc. Each of which are incorporated as reference in their entirety.
The present invention provides a novel purification process of antibodies by employing the conventional column chromatography techniques in a unique manner to obtain a highly purified preparation of desired antibody while removing the process- and product-related impurities especially the protein aggregates. We herein disclose such a purification process.